Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

p38/synaptophysin is a membrane protein present in clear (synaptic) vesicles of neurons and endocrine ceHs [1-4]. From the amino acid sequence deduced from cDNAs encoding p38/synaptophysin, a model with several membrane spanning polypeptide segments and a carboxy-terminal protein domain exposed to the cytoplasmic surface has been constructed [5-7]. The function of p38/synaptophysin is not known. It has been suggested to form a transmembrane channel for ions, or to interact with cytoplasmic factors via its cytoplasmic domain [7]. Since synaptophysin binds Ca2 +, it may also play a role in the release of neurotransmitters stored in clear (synaptic) vesicles [3]. Recently it has been reported [8] that p38/synaptophysin also occurs in hormone containing large dense core vesicles. This would imply that p38/synaptophysin could fulfill similar functions as described above in chromaffin and other secretory ceHs containing large dense core vesicles. In dear (synaptic) vesicles p38/synaptophysin constitutes 7.51Jfo of the vesicle membrane proteins [I]. The amount of p38/synaptophysin in large dense core vesides is not known. Here we report on the quantification of p38/synaptophysin in highly purified chromaffin secretory veside

The toxicity of chlorpyrifos towards differentiating mouse N2a neuroblastoma cells

The aim of this work was to study the effects of chlorpyrifos (CPF) on the outgrowth of axons by differentiating mouse N2a neuroblastoma cells. This was achieved by morphological, Western blotting and enzymatic analyses of cells induced to differentiate in the presence and absence of CPF added either at the same time (co-differentiation) or 16 h after (post-differentiation) the induction of cell differentiation. The outgrowth of axon-like processes was impaired following 4 or 8 h exposure to CPF in both co- and post-differentiation experiments. Western blotting analysis revealed reduced levels of neurofilament heavy chain (NF-H) following 8 h of exposure but no significant effect at 4 h under both co- and post-differentiation conditions. By contrast, levels of the heat shock protein HSP-70 were raised at both time points, but only in co-differentiation experiments. Neuropathy target esterase (NTE) activity was lower than controls following 4 or 8 h of exposure under co-differentiation conditions, but not under any post-differentiation conditions. The results suggest that the inhibition of axon production and maintenance by CPF in differentiating N2a cells may involve multiple targets, which are different under co- and post-differentiation conditions

Reduced tubulin tyrosination as an early marker of mercury toxicity in differentiating N2a cells

The aims of this work were to compare the effects of methyl mercury chloride and Thimerosal on neurite/process outgrowth and microtubule proteins in differentiating mouse N2a neuroblastoma and rat C6 glioma cells. Exposure for 4 h to sublethal concentrations of both compounds inhibited neurite outgrowth to a similar extent in both cells lines compared to controls. In the case of N2a cells, this inhibitory effect by both compounds was associated with a fall in the reactivity of western blots of cell extracts with monoclonal antibody T1A2, which recognises C-terminally tyrosinated α-tubulin. By contrast, reactivity with monoclonal antibody B512 (which recognises total α-tubulin) was unaffected at the same time point. These findings suggest that decreased tubulin tyrosination represents a neuron-specific early marker of mercury toxicity associated with impaired neurite outgrowth

METHOTREXATE ACTION IN RHEUMATOID ARTHRITIS: STIMULATION OF CYTOKINE INHIBITOR AND INHIBITION OF CHEMOKINE PRODUCTION BY PERIPHERAL BLOOD MONONUCLEAR CELLS

This open label study examines whether methotrexate (MTX) treatment modulates ex vivo synthesis of interleukin-1 receptor antagonist (IL-lra), soluble tumour necrosis factor receptors(sTNFR p55 and p75), interleukin-1β (IL-1β), tumour necrosis factor α(TNF-α), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by peripheral blood mononuclear cells (PBMC} and whether changes reflect clinical response. Significant stimulation of IL-lra and sTNFR p75 as well as inhibition of IL-8 production of PBMC were associated with clinical improvement observed in patients treated with MTX. When defining the characteristics of patients at study entry retrospectively in responders and non-responders a significantly lower ratio of IL-lra :IL-1β production before and its increase upon treatment was associated with clinical response in 13 patients compared to five patients not responding to MTX. In addition, clinical improvement was associated with decreased synthesis of IL-1β, TNF-α and IL-8 induced by bacterial lipopolysaccharide, IL-1α and IL-1β in PBMC in vitro. These findings suggest that MTX therapy reverses the inflammatory type of rheumatoid arthritis (RA) blood mononuclear cells by stimulating cytokine inhibitor production while inhibiting inflammatory cytokine release at the same time. This may explain the powerful anti-inflammatory properties of low-dose MTX as observed in most RA patients. Pretreatment determination of the IL-lra: IL-1β ratio in PBMC may be predictive with regard to a favourable therapeutic response and therefore may be useful for the selection of RA patients to be treated with MT

Characterization of Pulmonary Metastases in Children With Hepatoblastoma Treated on Children\u27s Oncology Group Protocol AHEP0731 (The Treatment of Children With All Stages of Hepatoblastoma): A Report From the Children\u27s Oncology Group.

Purpose To determine whether the pattern of lung nodules in children with metastatic hepatoblastoma (HB) correlates with outcome. Methods Thirty-two patients with metastatic HB were enrolled on Children\u27s Oncology Group Protocol AHEP0731 and treated with vincristine and irinotecan (VI). Responders to VI received two additional cycles of VI intermixed with six cycles of cisplatin/fluorouracil/vincristine/doxorubicin (C5VD), and nonresponders received six cycles of C5VD alone. Patients were imaged after every two cycles and at the conclusion of therapy. All computed tomography scans and pathology reports were centrally reviewed, and information was collected regarding lung nodule number, size, laterality, timing of resolution, and pulmonary surgery. Results Among the 29 evaluable patients, only 31% met Response Evaluation Criteria in Solid Tumors (RECIST) for measurable metastatic disease. The presence of measurable disease by RECIST, the sum of nodule diameters greater than or equal to the cumulative cohort median size, bilateral disease, and ≥ 10 nodules were each associated with an increased risk for an event-free survival event ( P = .48, P = .08, P = .065, P = .03, respectively), with nodule number meeting statistical significance. Ten patients underwent pulmonary resection/metastasectomy at various time points, the benefit of which could not be determined because of small patient numbers. Conclusion Children with metastatic HB have a poor prognosis. Overall tumor burden may be an important prognostic factor for these patients. Lesions that fail to meet RECIST size criteria (ie, those \u3c 10 mm) at diagnosis may contain viable tumor, whereas residual lesions at the end of therapy may constitute eradicated tumor/scar tissue. Patients may benefit from risk stratification on the basis of the burden of lung metastatic disease at diagnosis

Differential expression of synaptophysin and synaptoporin during pre- and postnatal development of the hippocampal network

The closely related synaptic vesicle membrane proteins synaptophysin and synaptoporin are abundant in the hippocampal formation of the adult rat. But the prenatal hippocampal formation contains only synaptophysin, which is first detected at embryonic day 17 (E17) in perikarya and axons of the pyramidal neurons. At E21 synaptophysin immunoreactivity extends into the apical dendrites of these cells and in newly formed terminals contacting these dendrites. The transient presence of synaptophysin in axons and dendrites suggests a functional involvement of synaptophysin in fibre outgrowth of developing pyramidal neurons. Synaptoporin expression parallels the formation of dentate granule cell synaptic contacts with pyramidal neurons: the amount of hippocampal synaptoporin, determined in immunoblots and by synaptoporin immunostaining of developing mossy fibre terminals, increases during the first postnatal week. Moreover, in the adult, synaptoporin is found exclusively in the mossy fibre terminals present in the hilar region of the dentate gyrus and the regio inferior of the cornu ammonis. In contrast, synaptophysin is present in all synaptic fields of the hippocampal formation, including the mossy fibre terminals, where it colocalizes with synaptoporin in the same boutons. Our data indicate that granule neuron terminals differ from all other terminals of the hippocampal formation by the presence of both synaptoporin and synaptophysin. This difference, observed in the earliest synaptic contacts in the postnatal hippocampus and persisting into adult life, suggests distinct functions of synaptoporin in these nerve terminals

An amphitropic cAMP-binding protein in yeast mitochondria

ABSTRACT: We describe the first example of a mitochondrial protein with a covalently attached phos-phatidylinositol moiety acting as a membrane anchor. The protein can be metabolically labeled with both stearic acid and inositol. The stearic acid label is removed by phospholipase D whereupon the protein with the retained inositol label is released from the membrane. This protein is a cAMP receptor of the yeast Saccharomyces cereuisiae and tightly associated with the inner mitochondrial membrane. However, it is converted into a soluble form during incubation of isolated mitochondria with Ca2+ and phospholipid (or lipid derivatives). This transition requires the action of a proteinaceous, N-ethylmaleimide-sensitive component of the intermembrane space and is accompanied by a decrease in the lipophilicity of the cAMP receptor. We propose that the component of the intermembrane space triggers the amphitropic behavior of the mitochondrial lipid-modified CAMP-binding protein through a phospholipase activity. Only in recent years specific fatty acids have been recog-nized to play important roles in the association of proteins with membranes. Both noncovalent and covalent interactions be-tween fatty acids and proteins have been reported. Among the latter are GTP-binding proteins (Molenaar et al., 1988)

Identification of the protein kinases Pyk3 and Phg2 as regulators of the STATc-mediated response to hyperosmolarity

Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In Dictyostelium, STATc is a key regulator of the transcriptional response to hyperosmotic stress. Its phosphorylation and consequent activation is controlled by two signaling branches, one cGMP- and the other Ca(2+)-dependent, of which many signaling components have yet to be identified. The STATc stress signalling pathway feeds back on itself by upregulating the expression of STATc and STATc-regulated genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of STATc phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of STATc and STATc dependent genes was disturbed in pyk3(-), phg2(-), and pyk3(-)/phg2(-) cells. The absence of Pyk3 and/or Phg2 resulted in diminished or completely abolished increased transcription of STATc dependent genes in response to sorbitol, 8-Br-cGMP and the Ca(2+) liberator BHQ. Also, phospho-STATc levels were significantly reduced in pyk3(-) and phg2(-) cells and even further decreased in pyk3(-)/phg2(-) cells. The reduced phosphorylation was mirrored by a significant delay in nuclear translocation of GFP-STATc. The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits STATc, is inhibited by stress-induced phosphorylation on S448 and S747. Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3 is involved in the phosphorylation of PTP3 on S747. In pull-down assays Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2(-) cells. We show that Phg2 and an, as yet unknown, S448 protein kinase are responsible for PTP3 phosphorylation and hence its inhibition, and that Pyk3 is involved in the regulation of STATc by either directly or indirectly activating it. Our results add further complexities to the regulation of STATc, which presumably ensure its optimal activation in response to different environmental cues